Integrated genetic and physical genome maps are extremely valuable for map-based gene isolation, comparative genome analysis and as sources of sequence-ready clones for genome sequencing projects. The effect of the availability of an integrated map of physical and genetic markers of a species for genome research is enormous. Integrated maps allow for precise and rapid gene mapping and mapping of all microsatellite loci and other application such as SNA marker based gene manipulation. Various methods have been developed for assembling physical maps of genomes of varying complexity. One of the better characterised approaches use restriction enzymes to generate large numbers of DNA fragments from genomic subclones (Brenner et al., Proc. Natl. Acad. Sci., (1989), 86, 8902-8906; Gregory et al., Genome Res. (1997), 7, 1162-1168; Marra et al., Genome Res. (1997), 7, 1072-1084). These fingerprints are compared to identify related clones and to assemble overlapping clones in contigs. The utility of fingerprinting for ordering a complex genome is limited, however, due to variation in DNA migration from gel to gel, the presence of repetitive DNA sequences, unusual distribution of restriction sites and skewed clone representation. Moreover, fingerprinting alone, unless combined with other methods, does not link genomic clones directly to genetic maps. Therefor most high quality physical maps of complex genomes have been constructed using a combination of fingerprinting and PCR-based or hybridisation based methods.
Selective restriction fragment amplification or AFLP is known, for instance from the European patent application 0 534 858 and U.S. Pat. No. 6,045,994 by applicant and from an article by Vos et al. Nucleic Acids Research (1995), 23, 4407-4414, incorporated herein by reference. In general, AFLP comprises the steps of:                (a) digesting a nucleic acid, in particular a DNA or a cDNA, with one or more specific restriction endonucleases, to fragment said DNA into a corresponding series of restriction fragments;        (b) ligating the restriction fragments thus obtained with at least one double-stranded synthetic oligonucleotide adapter, one end of which is compatible with one or both of the ends of the restriction fragments, to thereby produce tagged restriction fragments of the starting DNA;        (c) contacting said tagged restriction fragments under hybridising conditions with at least one oligonucleotide primer;        (d) amplifying said tagged restriction fragments hybridised with said primers by PCR or a similar technique so as to cause further elongation of the hybridised primers along the restriction fragments of the starting DNA to which said primers hybridised; and        (e) identifying or recovering the amplified or elongated DNA fragment thus obtained.        
The amplified DNA-fragments thus obtained can then be analysed and/or visualised, for instance by means of gel-electrophoresis. This provides a genetic fingerprint showing specific bands corresponding to the restriction fragments which have been linked to the adapter, have been recognised by the primer, and thus have been amplified during the amplification step. The fingerprint thus obtained provides information on the specific restriction site pattern of the starting DNA, and thus on the genetic make-up of the organism from which said DNA has been derived.
AFLP can therefore be used to identify said DNA; to analyse it for the presence of specific restriction site patterns, restriction fragment length polymorphisms (RFLPs) and/or specific genetic markers (so-called “AFLP-markers”), which may be indicative of the presence of certain genes or genetic traits; or for similar purposes, for instance by comparing the results obtained to DNA-samples of known origin or restriction pattern, or data thereon. AFLP is eminently suited to characterise genetic markers by means of one or more of the AFLP fragments thus visualised.
The primers used in AFLP are such that they recognise the adapter and can serve as a starting point for the polymerase chain reaction. To this end, the primers must have a nucleotide sequence that can hybridise with (at least part of) the nucleotide sequence of the adapter adjacent to the 3′ end of the restriction fragment to be amplified. The primers can also contain one or more further bases (called “selective bases”) at the 3′-end of their sequence, for hybridisation with any complementary base or bases at the 3′-end of the adapter ligated restriction fragment. Located between the part of the primer that hybridises to the adapter and the selective bases that hybridise to the restriction fragment, the primer may contain a section that is capable of hybridising to the remains of the restriction site. Thus, in general an AFLP primer has the following structure: adapter complementary part-restriction site remains complementary part-selective bases. The adapter complementary part-restriction site remains complementary part is generally depicted as the ‘constant sequence’ of the AFLP primer and the selective bases as the ‘variable sequence’.
As, of all the adapter-ligated restriction fragments present in the mixture, only those fragments that contain bases complementary to the selective bases will subsequently be amplified, the use of these “selective” primers will reduce the total amount of bands in the final fingerprint, thus making the fingerprint more clear and more specific. Also, the use of different selective primers (i.e. different variable sequence) will generally provide different fingerprints, which can also be used as a tool for the purposes of identification or analysis.
The selective nucleotides are complementary to the nucleotides in the adapter-ligated restriction fragments that are located adjacent to the constant primer sequence.
Primers containing selective nucleotide are denoted as +N primers, in which N stands for the number of selective nucleotides present at the 3′-end of the primer. N is preferably selected from amongst A, C, T or G.
N may also be selected from amongst various nucleotide alternatives, i.e. compounds that are capable of mimicking the behaviour of ACTG-nucleotides but in addition thereto have other characteristics such as the capability of improved hybridisation compared to the ACTG-nucleotides or the capability to modify the stability of the duplex resulting from the hybridisation. Examples thereof are peptide nucleic acids (PNAs), locked nucleic acids (LNAs), inosine etc. When the amplification is performed with more than one primer, such as with PCR using two primers, one or both primers can be equipped with selective nucleotides. The number of selective nucleotides may vary, depending on the species or on other particulars determinable by the skilled man. In general the number of selective nucleotides is not more than 10, but at least 5, preferably 4, more preferably 3, most preferred 2 and especially preferred is 1 selective nucleotide.
A +1 primer thus contains one selective nucleotide, a +2 primer contains 2 selective nucleotides etc. A primer with no selective nucleotides (i.e. a conventional primer) can be depicted as a +0 primer (no selective nucleotides added). When a specific selective nucleotide is added, this is depicted by the notion +A or +C etc.
By amplifying a set adapter ligated restriction fragments with a selective primer, a subset of adapter-ligated restriction fragments is obtained, provided that the complementary base is present at the appropriate position in the restriction fragment. Using a +1 primer, for example, the complexity (and the number of visualised fragments) of the amplified mixture is reduced by a factor 4 compared to a amplification with a non-selective primer (a+0 primer) Higher reductions can be achieved by using primers with multiple selective nucleotides, i.e. 16 fold reduction of the original multiplex ration is obtained with 2 selective nucleotides etc.
As AFLP provides amplification of both strands of a double stranded starting DNA, AFLP advantageously allows for exponential amplification of the fragment, i.e. according to the series 2, 4, 8, 16, etc. Also, AFLP requires no prior knowledge of the DNA sequence to be analysed, nor prior identification of suitable probes and/or the construction of a gene library from the starting DNA.
For a further description of AFLP, its advantages, its embodiments, as well as the techniques, enzymes, adapters, primers and further compounds and tools used therein, reference is made to EP-0 534 858, and to Vos et al. Nucleic Acids Research (1995), 23, 4407-4414 both publications are incorporated herein by reference. Also, in the description hereinbelow, the definitions given in paragraph 5.1 of EP-0 534 858 will be used, unless indicated otherwise.
The potential of AFLP as a technology for the integration of physical and genetic maps has been recognised before. Klein et al. in Genome Research, (2000), 10, 798-807 have described the use of AFLP in the integration of physical and genetic maps of Sorghum. The method of Klein et al. comprises generating AFLP fingerprints using +3/+3 selective primers of all individual BAC clones in a library. The method further comprises the generation of pools of clones. The pools are also analysed by AFLP fingerprinting albeit using different restriction enzymes and otherwise other circumstances compared to the generation of the AFLP fingerprints of all the individual BACs. The use of different enzyme combinations renders the method difficult. The method of Klein et al. although feasible, is complex and laborious and comes with several other disadvantages. One of them is that the method is unsuitable for positioning non-polymorphic markers on the integrated map.
It is a goal of the present invention to provide for an improved method for the integration of physical and genetic maps. It is a further goal of the present invention to provide for an improved method based on AFLP. It is yet a further goal of the invention to provide for a high throughput method for the integration of physical and genetic maps that results in maps of improved quality such as measured by increased marker densities or as a result of more reliable contig generation.